<R. Gu 4/6/2005
24 well dishes
Millicell 0.4 µm inserts (Millipore PICM01250) - coated with fibronectin
Fixative: 4% paraformaldehyde in PBS (pH 7.6)
Rabbit anti-GFP (Molecular Probes, A-11122)
FITC-goat anti-rabbit IgG (Jackson ImmunoResearch, 111-096-006)
Biotinylated anti-BrdU (AbCam, ab2284-125)
Streptavidin-Alexa Flour 555 (Invitrogen/Molecular Probes, S21381)
DAPI Fluorescent mounting medium (Vectashield, Vector Labs H-1200)
Glass microscope slides and cover slips
- Coat Millicell inserts with fibronectin as described by Millipore.
- Cultivate tissue at 37ºC on fibronectin coated well inserts in a 24-well dish.
- Remove well inserts and cut out mesh insert with a scalpel, forceps and dissecting microscope
- Place inserts in a 2.0 mL Eppendorf tube with 1 mL of blocking agent (10% goat serum, 1% BSA in PBS). Gently flick tube to mix.
- Remove solution and wash 3x by adding 1 mL PBS, 3' each.
- Add 0.3 mL rabbit anti-GFP (1:200 v/v, plus 1% goat serum in PBS) and incubate 1 hr. at room temp. Wash 3x in PBS.
- Post-fix in 1 mL 4% paraformaldehyde x15', room temp. Wash 3x in PBS.
- Denature DNA with 1 mL of 2 N HCl x30'. Wash 3x in PBS.
- Block in 1 mL 10% Horse serum, x30' at room temp. Wash 1x in PBS.
- Add 0.3 mL Biotinylated anti-BrdU (1:300, with 1% horse serum in PBS). Incubate at 4ºC o.n. or 2 hr. at room temp. Wash 3x in PBS.
- Dilute 1 µL fresh streptavidin-Alexa Flour 555 in 1 mL PBS (1:1,000). Spin the dilute antibody at high speed for 30' to remove particulates. Add supernatant to specimen and incubate 30' at room temp.
- Remove antibody and add 1 mL PBS. O.K. to store at 4ºC.
- Use a scalpel to remove membrane from well inserts. Spot 25-50 µL of DAPI mounting medium onto a glass slide. Place 1-2 membranes on the DAPI medium. Use a dissecting microscope to be sure to place the tissue side up. Cover with a 24-40 mm glass slip. Paint the edges with clear nail polish. View with immunoflourescence. Store in the dark at 4ºC.